PROJECT DESCRIPTION The sterile alpha motif (SAM) and HD-domain containing protein 1 (SAMHD1) plays important roles in the intrinsic immunity defense and potently blocks post-entry infection of HIV-1 early at a stage of reverse transcription in non-dividing human immune cells. Mutations in SAMHD1 are associated with human diseases including chronic lymphocytic leukemia and the autoimmune condition Aicardi-Goutieres syndrome (AGS). SAMHD1 is a deoxyribonucleotide triphosphohydrolase (dNTPase). The dNTPase activity of SAMHD1 is critical for cellular dNTP regulation in mammalian cells. The antiviral activity of SAMHD1 is negatively modulated in dividing cells by phosphorylation at residue T592. Murine SAMHD1 (mSAMHD1), which is 72% sequence identical to human SAMHD1 (hSAMHD1), also restricts HIV-1 through its dNTPase activity. Interestingly, there are two isoforms of mSAMHD1, with the antiviral activity of isoform 1 (iso1) regulated by phosphorylation at residue T634, while isoform 2 (iso2) lacks this site. Despite intensive investigation, the mechanism of viral restriction by SAMHD1, particularly the function of the SAM domain, still remains unclear and controversial. The overall goal of the proposed research is to further establish the biochemical and structural basis by which full-length SAMHD1 inhibits retroviral infection and the regulatory mechanisms of SAMHD1 activities. We hypothesize that both the SAM domain and phosphorylation modulate SAMHD1 dNTPase and antiviral activities. To achieve our goals, we will use a combination of techniques including biochemistry, biophysics, structural and molecular biology, cell biology, and virology to elucidate the regulation of the enzyme activities and antiviral functions. We propose the following specific aims: Aim 1. To elucidate the activities of mouse SAMHD1 in retroviral restriction. Aim 2. To delineate the regulatory function of the SAM domain in SAMHD1. Aim 3. To establish a comprehensive description of SAMHD1 regulation by phosphorylation. The success of the proposed work will greatly advance the mechanistic understanding of SAMHD1, an important host immune strategy to combat HIV. It will establish the relevance of SAMHD1 enzymatic activities in its retroviral restriction and other cellular functions. Understanding the functional attributes of the mouse enzyme will provide novel information to improve the mouse model for investigating the replication of retroviruses in vivo. A comprehensive understanding of the regulation and modulation of the enzymatic activities of SAMHD1 is crucial for assessing its potential as a therapeutic target for AIDS, certain cancers and autoimmune diseases.